Drosophila Melanogaster Mariya V Zhukova, Elena Kiseleva*

Zhukova and Kiseleva BMC Microbiology 2012, 12(Suppl 1):S15 http://www.biomedcentral.com/1471-2180/12/S1/S15

RESEARCH Open Access The virulent Wolbachia strain wMelPop increases the frequency of apoptosis in the female germline cells of Drosophila melanogaster Mariya V Zhukova, Elena Kiseleva*

Abstract Background: Wolbachia are bacterial endosymbionts of many arthropod species in which they manipulate reproductive functions. The distribution of these bacteria in the Drosophila ovarian cells at different stages of oogenesis has been amply described. The pathways along which Wolbachia influences Drosophila oogenesis have been, so far, little studied. It is known that Wolbachia are abundant in the somatic stem cell niche of the Drosophila germarium. A checkpoint, where programmed cell death, or apoptosis, can occur, is located in region 2a/2b of the germarium, which comprises niche cells. Here we address the question whether or not the presence of Wolbachia in germarium cells can affect the frequency of cyst apoptosis in the checkpoint. Results: Our current fluorescent microscopic observations showed that the wMel and wMelPop strains had different effects on female germline cells of D. melanogaster. The Wolbachia strain wMel did not affect the frequency of apoptosis in cells of the germarium. The presence of the Wolbachia strain wMelPop in the D. melanogasterw1118 ovaries increased the number of germaria where cells underwent apoptosis in the checkpoint. Based on the appearance in the electron microscope, there was no difference in morphological features of apoptotic cystocytes between Wolbachia-infected and uninfected flies. Bacteria with normal ultrastructure and large numbers of degenerating bacteria were found in the dying cyst cells. Conclusions: Our current study demonstrated that the Wolbachia strain wMelPop affects the egg chamber formation in the D. melanogaster ovaries. This led to an increase in the number of germaria containing apoptotic cells. It is suggested that Wolbachia can adversely interfere either with the cystocyte differentiation into the oocyte or with the division of somatic stem cells giving rise to follicle cells and, as a consequence, to improper ratio of germline cells to follicle cells and, ultimately, to apoptosis of cysts. There was no similar adverse effect in D. melanogaster Canton S infected with the Wolbachia strain wMel. This was taken to mean that the observed increase in frequency of apoptosis was not the general effect of Wolbachia on germline cells of D. melanogaster,it was rather induced by the virulent Wolbachia strain wMelPop.

Background cells remain undamaged [3,4]. Apoptosis is a complex Apoptosis, a form of programmed cell death, is a pro- process whereby a proteolytic cascade of caspases is cess needed for normal development and maintenance activated in cells [5]. of tissue homeostasis in multicellular organisms [1,2]. The occurrence of apoptosis is a feature of female Cells undergoing apoptosis show characteristic changes, germline development common to vertebrate and inver- such as chromatin and cytoplasm condensation, chro- tebrate species [6,7]. In the Drosophila melanogaster mosomal DNA fragmentation, breaking up of nuclei and ovaries, there are two checkpoints where programmed then of cells into fragments called apoptotic bodies cell death occurs. One is in the germarium (region 2a/ [3,4]. Individual cells apoptose, while the neighboring 2b), where apoptosis probably regulates the proper ratio of germline cells to follicle cells [8]. The other check- * Correspondence: [email protected] point is located in the vitellarium (stages 7-8 of oogen- Institute of Cytology and Genetics, Siberian Branch of the Russian Academy esis) [9]. The number of egg chambers undergoing of Sciences, Novosibirsk, 630090 Russia

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apoptosis increased in D. melanogaster fed a diet lacking from D. melanogaster Canton S. In contrast, the number protein [8], under the effect of 900-MHz and 1800-MHz of germaria containing apoptotic cells in the checkpoint radiation [10], and after exposure to chemical agents was considerably increased in the wMelPop-infected [11]. The normal development of mature egg is consis- flies as compared with their uninfected counterparts. tently associated with apoptosisof15nursecellsinthe Thus, evidence was obtained indicating that the virulent egg chamber [12]. It is noteworthy that apoptosis and Wolbachia strain wMelPop has an effect on the fate of autophagy coexist at all the above mentioned stages of germline cells during D. melanogaster oogenesis. oogenesis in D. melanogaster [13,14]. It has been also hypothesized that the apoptotic pro- Results cess had a symbiotic origin [15]. In terms of the endo- Frequency of apoptosis in germaria from ovaries of the symbiotic theory, mitochondria, which play a major role uninfected and Wolbachia-infected D. melanogaster at the early stages of apoptosis, evolved from the free- Two parts are distinguished in the Drosophila ovariole: living prokaryotes [5]. One of the symbionts may be the germarium made up of four regions (1, 2a, 2b, 3) involved in the regulation of apoptosis in partner cells. and the vitellarium (Figure 1A, B) [27,28]. The region To illustrate, extracellular parasites, particularly such 2a/2b, where apoptosis can occur, contains 16-cell cysts, worms as filarial nematodes, schistosomes and the ces- somatic stem cells (SSCs), which contact with the tode Taenia crassiceps, are able to induce apoptosis in somatic stem cell niche (SSCN) and follicle cells (Figure host immune cells [16]. Bacterial pathogens (Chlamydia, 1B). Cell death in this region of the germarium was Neisseria, Legionella pneumophila) can either block or detected by two methods, acridine orange (AO)-staining induce apoptosis in host cells, depending on the stage of and TUNEL assay. Fluorescence microscopy of AO- infection [17,18]. At the early stage of infection, bacteria stained ovarioles demonstrated that apoptotic cells were replicate in the host cell, using different mechanisms to located as large yellow or orange spots in region 2a/2b prevent apoptosis. At the late stages of infection, the of the germarium from D. melanogaster (Figure 2A, C, bacteria induce apoptosis in the host cell, thereby facili- E, G). Germaria containing no apoptotic cells fluoresced tating egress and ensuring infection of neighboring cells. homogeneous green (Figure 2B, D, F, H). It should be Wolbachia associated with various hosts in which it noted that wMel- and wMelPop-infected flies, besides manipulates viability and reproduction causing parthe- bright spots in region 2a/2b (Figure 2C, G), showed nogenesis, feminization, male killing and cytoplasmic weak punctuate fluorescence both in regions 2a/2b and incompatibility, provides a unique model for studying 1ofthegermarium(Figure2C,D,G,H).Suchfluores- mechanisms of symbiont interactions [19,20]. The Wol- cent puncta were not observed following TUNEL, bachia strain wMel is widely spread in natural popula- thereby indicated that they were not caused by tions of D. melanogaster [21,22]; in contrast, wMelPop apoptosis. has been detected in a laboratory stock of D. melanoga- The percentage of germaria containing apoptotic cells ster [23]. It is possibly not encountered in nature. In D. was 41.8±4.1% in the uninfected D. melanogasterw1118T melanogaster, the wMelPop strain reduces lifespan, pro- maintained on standard food, whereas it increased to liferating widely in the brain, muscle and retina cells 70.6±5.3% in the wMelPop-infected flies (Figure 2I). [23]. In certain insect species, the presence of Wolba- Analysis performed with the wMel-infected D. melano- chia is required for oogenesis [24]. Removal of the Wol- gaster Canton S revealed no significant differences from bachia strain wAtab3 from parasitic wasps Asobara their uninfected counterparts (Figure 2I, Table 1).The tabida resulted in copious apoptosis of the egg cham- next step was to exclude the possible effect of insuffi- bers in the ovarioles and led to sterility [25]. The cient nutrition on the current results. To do so, we con- mechanisms whereby the endosymbiont Wolbachia ducted experiments in which flies were raised on rich impacts apoptosis in host cells have been poorly studied. food source taking into account that it decreases the Preferential infection and high accumulation of Wolba- number of germaria containing apoptotic cells [8,29]. chia in region 2a of the germarium [26] where the We found that rich food causes a decrease in the rela- checkpoint is located in Drosophila was thought-provok- tive proportion of apoptotic germaria in both w1118T ing. We raised the question: Can bacteria Wolbachia in and w1118 flies; however, the difference between these region 2a of the germarium affect the frequency of two groups was significant (Figure 2I, Table 1). The per- apoptosis there? Using fluorescence and transmission centage of germaria containing apoptotic cells did not electron microscopy we compared germaria from ovaries change under the effect of rearing D. melanogaster Can- of two D. melanogaster stocks infected with either the ton S on different food. Based on analysis of apoptotic wMel or wMelPop strains with germaria from two unin- cell death by TUNEL, three groups of germaria were fected counterparts. It was established that the presence distinguished: TUNEL-negative, TUNEL-positive with 1- of wMel did not alter apoptosis frequency in germaria 2 distinct puncta in region 2a/2b and TUNEL-positive Zhukova and Kiseleva BMC Microbiology 2012, 12(Suppl 1):S15 Page 3 of 12 http://www.biomedcentral.com/1471-2180/12/S1/S15

Figure 1 A schematic representation of an ovariole of D. melanogaster. A, an ovariole of D. melanogaster consisting of the germarium (g) and the vitellarium. B, a detailed scheme of the germarium structure composed of regions 1, 2a, 2b, 3. The checkpoint is framed (red). C, a 16- cell cyst; SSCN, a somatic stem cell niche; SSC, a somatic stem cell; FC, a follicle cell. with a cluster of bright spots (Additional file 1). There consisted of nuclei that exhibited numerous invagina- was no evidence for variation in the frequency of apop- tions, protrusions, and cytoplasm rich in organelles (Fig- tosis between wMel-infected (Canton S) and uninfected ure 3C, D, Additional file 2). Our ultrastructural data (Canton ST) flies (Table 2; c2=1.3, df=1, P=0.25); how- for germarium cells of the uninfected and the Wolba- ever, there was evidence for a difference in the fre- chia-infected flies allowed us to identify cysts in region quency of apoptosis between the w1118T and w1118 flies 2a/2b showing characteristic features of apoptotic death (Table 2; c2=25.3, df=1, P<0.0001). The total percentage (Figure 4 and Additional file 3). The cytoplasm was of germaria containing apoptotic cells in D. melanoga- more electron-dense in such cystocytes, some mitochon- ster agreed well with the one obtained with AO-staining. dria became markedly swollen (Figs. 4A and Additional Thus, TUNEL confirmed the results of AO-staining. file 3A). The matrix of mitochondria was light and just a few small cristae were discerned at the periphery (Figs. Ultrastructure of germaria from ovaries of the uninfected 4B and Additional file 3B). We observed also cells with and the Wolbachia-infected D. melanogaster electron-dense cytoplasm, which had lost contact with For an ultrastructural analysis of germarium cells, we their neighboring cells (Additional file 3C). In such cells, first chose under the light microscope those longitudinal chromatin appeared condensed in apoptotic nuclei and sections that enabled us to define region 2a/2b of the the lumen of the nuclear envelope was dilated (Figs. 4C germarium (Figure 3A, B, red brackets). Cyst cells in and Additional file 3C). At the last stage of apoptosis, region 2a/2b were interconnected by ring canals and cells disaggregated into large and small fragments, or Zhukova and Kiseleva BMC Microbiology 2012, 12(Suppl 1):S15 Page 4 of 12 http://www.biomedcentral.com/1471-2180/12/S1/S15

Figure 2 Visualisation of acridine orange (AO)- and TUNEL-stained germarium cells of D. melanogaster. A, C, E, G, germaria containing apoptotic cells in region 2a/2b from 5 day-old uninfected (A, E) and Wolbachia-infected (C, G) females (AO staining). B, D, F, H, germaria not containing apoptotic cells from the same fly stocks (AO staining). Arrows indicate small punctate AO-staining in regions 1 and 2a/2b (C, D, G, H). I, relative proportion of germaria containing apoptotic cells from ovaries of the uninfected (w1118T, Canton ST) and Wolbachia-infected (w1118, Canton S) flies. The total number of examined germaria is indicated by blue number; bars show the average percentage per experiment ± s. e. m. J, L, germaria containing apoptotic cells in region 2a/2b in the wMelPop- and wMel-infected fly stocks, respectively (TUNEL). K, M, germaria not containing apoptotic cells from the same fly stocks. Region 2a/2b of the germarium is indicated by red brackets. Scale bars: 20 μm. apoptotic bodies, with characteristic electron-dense morphological appearance (5C, D and Additional file cytoplasm containing ribosomes, endoplasmic reticulum 4B). Some endosymbionts showed normal ultrastructural membranes, and frequently intact mitochondria (Figs. features: a three-layered envelope, a matrix with many 4D and Additional file 3D). ribosomes and dispersed chromatin. In contrast, most Analysis of germarium cystocytes of wMel- and wMel- bacteria were surrounded by a three-layered envelope, Pop-infected flies showed that individual bacteria were the matrix was of low electron density with a few ribo- distributed throughout all the cytoplasm, occasionally somes. Disrupting bacteria were also encountered. occurring as small groups (Figs 3D and Additional file These were not enclosed by an envelope, their matrix 2). Large accumulations of adjacent bacteria were was loose, light, devoid of ribosomes. The follicle cells observed in apoptotic cyst cells (Figure 5A, B, Addi- surrounding the cysts in region 2b of the germarium tional file 4). In these masses, the bacteria varied in showed a normal morphology and low levels of Wolba- chia with normal structure (Additional file 5). At the periphery of the germarium, fragments of degrad- Table 1 Details of statistical analysis (two-way ANOVA) ing cells were frequently seen in region 1, precisely Source of Canton S/Canton ST w1118/w1118T where AO-staining of the germaria from the Wolbachia- variation infected flies was punctate (Figure 2C, D, G, H). These % of total P % of total P value fragments were filled with multilayered membranes, variation value variation nuclear remnants, mitochondria, and bacteria with nor- Interaction 1,51 0,7065 0.74 0,4998 mal and abnormal morphology (Figure 6A-C, Additional Type of food 0,15 0,9045 9,23 0,0312 file 6). The cell organelles and bacteria were often Infection status 19,30 0,1998 63,68 P<0.0001 engulfed by autophagosomes. Besides bacteria with light Data of AO-staining of germaria from 4 fly stocks maintained at different food. matrix, like those in apoptotic cysts (Figure 6C, D), the Zhukova and Kiseleva BMC Microbiology 2012, 12(Suppl 1):S15 Page 5 of 12 http://www.biomedcentral.com/1471-2180/12/S1/S15

Table 2 Quantification of TUNEL staining in region 2a/2b of the germaria Fly stock TUNEL-negative TUNEL-positive Total % of TUNEL-positive Total number Distinct puncta Cluster of spots Canton ST 61.6% (133) 8.8% (19) 29.6% (64) 38.4% 216 Canton S 56.3% (134) 10.1% (24) 33.6% (80) 43.7% 238 w1118T 59.1% (111) 13.8% (26) 27.1% (51) 40.9% 188 w1118 34.6% (82) 14.3% (34) 51.1% (121) 65.4% 237 autophagosomes occasionally enclosed electron-dense has no effect on sequential passage of intracellular orga- bacteria-like structures 0.2-0.3 μm in diameter (Figure nelles through apoptosis. To revealthepossiblediffer- 6D, E) or similar smaller ones (Figure 6F). At the per- ences between the effect of the wMel and wMelPop iphery of the germaria, autophagosomes containing indi- strains on apoptosis in the germaria, additional morpho- vidual bacteria with normal morphology were observed metric analysis of the numberofapoptoticstructures (Figure 6G). and of Wolbachia density in the cystocytes is required.

Discussion Structural features of Wolbachia in apoptotic cysts This is, to our knowledge, the first study that demon- Wolbachia with matrix of moderate and low electron strated by using AO- and TUNEL staining an increase density in apoptotic cells in region 2a/2b of the germar- in the frequency of apoptosis in the germarium check- ium have been previously encountered in other types of point in wMelPop-infected D. melanogasterw1118.This D. melanogaster ovaries [34] and they presumably reflect increase is possibly caused by the specific effect of the different functional states of bacteria. Wolbachia with Wolbachia strain wMelPop, since it was not observed in disrupted envelopes and light matrix are possibly dying wMel-infected D. melanogaster Canton S. Our current bacteria in apoptotic cells. Such appearance has not electron microscopic observations allowed us to identify been observed in Wolbachia injured or killed by heat changes in Wolbachia morphology in apoptotic germline stress [35] and tetracycline [36]. The electron-dense bac- cells. teria-like structures at the periphery of region 1 of the germarium may be evidence of changes in dying Wolba- Morphological evidence of apoptosis in germarium cells chia. Large masses of structures of this kind resembling The ultrastructural features of apoptosis in the cyst cells the bacteria endospores have been found in the brain of higher eukaryotes have gained wide recognition. They cells of the wMelPop-infected D. melanogasterw1118[23]. include cytoplasmic and nuclear condensation (pykno- In our view, the electron-dense structures, which we sis); nuclear fragmentation (karyorrhexis); normal mor- revealed at the periphery of region 1 of the germarium, phological appearance of cytoplasmic organelles; an are presumably autophagosome encapsulated dying Wol- intact plasma membrane [3,4]. The ultrastructural bachia. A supporting line of evidence came from Wright changes we identified here in D. melanogaster cyst cells and Barr [37], who on the basis of their observations on are consistent with the above hallmarks. Furthermore, degenerating germaria cysts from mosquitoes Aedes scu- we revealed mitochondria of two types: intact morphol- tellaris suggested that these structures represented ogy in one type and markedly swollen with a few cristae degenerating Wolbachia. in the other. A similar heterogeneity of mitochondrial Cell fragments containing dying bacteria and autopha- ultrastructure has been observed during apoptosis in gosomesandappearingasnumeroussmallerpunctain granulose cells of Japanese quail (Coturnix coturnix regions 2a/2b and 1 of the germarium may represent japonica) [30], lymphocytes from leukemia patients [31], autophagy, not apoptosis. This appears plausible when and megakaryocytes from patients with idiopathic recalling that AO stains not only apoptotic cells, also thrombocytopenic purpura [32]. It has been suggested lysosomes [38]. TUNEL did not reveal such puncta in thattheswollenmitochondriareleasecytochromec, these regions. which activates a cascade of proteolytic reactions, while the normal ones retain their capacity for ATP synthesis, The possible role of the Wolbachia strain wMelPop in a process apoptosis requires [30,31,33]. According to programmed cell death in region 2a/2b of the germarium our qualitative analysis using EM, morphological evi- Our current estimates of apoptosis in region 2a/2b of dence of apoptosis was revealed in germline cells from the germarium from the ovaries of the uninfected D. uninfected flies and those infected with wMel and melanogasterw1118T raised on standard food are consis- wMelPop. Thus, there are reasons for inferring that the tent with those reported elsewhere [14]. It is of interest endosymbiont Wolbachia in D. melanogaster cystocytes that apoptosis level in the germaria decreased in D. Zhukova and Kiseleva BMC Microbiology 2012, 12(Suppl 1):S15 Page 6 of 12 http://www.biomedcentral.com/1471-2180/12/S1/S15

Figure 3 Visualisation of germarium cells in semi-thin and ultra-thin sections. A, B, longitudinal semi-thin sections of germaria stained with methylene blue. C, D, ultrastructure of cyst cells from the uninfected and the wMelPop-infected flies. Arrows point to bacteria; arrowheads denote ring canals between neighboring cells. Scale bars correspond to 10 μm (A, B) and 2 μm (C, D), respectively. melanogasterw1118T,butnotinD. melanogaster Canton wMelPop increased the percentage of germaria contain- ST after transfer to rich food. This may be indicative of ing apoptotic cells in D. melanogasterw1118 ovaries, while differences in sensitivity to changes in food composition wMel strain was without such an effect. The effect of between different fly stocks. AO- and TUNEL staining wMelPop on cystocytes in ovaries was observed in flies demonstrated that the virulent Wolbachia strain maintained on standard and rich food. Evidence was Zhukova and Kiseleva BMC Microbiology 2012, 12(Suppl 1):S15 Page 7 of 12 http://www.biomedcentral.com/1471-2180/12/S1/S15

Figure 4 Morphology of apoptotic cystocytes in region 2a/2b of the germarium from the wMelPop-infected D. melanogasterw1118. A, swollen mitochondria (black arrows) in the cytoplasm of cyst cells. White arrows indicate bacteria. B, a fragment of a cyst cell with two mitochondria: one is normal, the other is swollen with the matrix of low electron density and the disintegrated cristae. C, a cyst cell, the cytoplasm appears dense, the nucleus is pyknotic. D, apoptotic bodies (ab) containing intracellular organelles. Scale bars: 1 μm. provided that the effect of Wolbachia on D. melanoga- microtubule-organizing center forms [39,40]. Wolbachia ster is not general, being rather specific to the patho- distribution is dependent upon microtubules during genic strain wMelPop. oogenesis and bacteria show mislocalization in the egg What pathways may be envisaged for the Wolbachia chambers treated with colchicine which causes depoly- strain wMelPop caused increase in the number of ger- merization of microtubules [41]. Evidence has been maria whose cysts undergo apoptosis? On the one hand, obtained indicating that Wolbachia areevenlydistribu- bacteria may have a direct effect on germline cells (Fig- ted throughout the oocyte and nurse cells during stages ure 7A, B). In fact, one of 16 cyst cells becomes the 1-2 of oogenesis, becoming concentrated at the oocyte oocyte, the other 15 differentiate into nurse cells in anterior during stages 3-6 [41]. With this in mind, the region 2a of the germarium. This is associated with high levels of Wolbachia in cystocytes during differen- transport of 15 centrioles into the pre-oocyte, where the tiation into oocyte and nurse cells in region 2a of the Zhukova and Kiseleva BMC Microbiology 2012, 12(Suppl 1):S15 Page 8 of 12 http://www.biomedcentral.com/1471-2180/12/S1/S15

Figure 5 Ultrastructure of the Wolbachia strain wMelPop in apoptotic cystocytes in region 2a/2b of the germarium. A, B, Wolbachia accumulations in apoptotic cyst cells, low magnification view. C,D, bacteria framed in panels A, B depicted at higher magnification. Bacteria showing normal morphology (arrows), bacteria with matrix of low electron density (white arrowheads), bacteria with matrix of low electron density and disrupted cell wall (black arrowheads) in the cytoplasm of dying cysts. Scale bars: 2 μm. germarium may possibly lead to impairment at the undergo apoptotic death. Judging from our current data, structural and/or molecular level, the cyst may undergo the ultrastructural appearance of follicle cells in region apoptosis as a consequence (Figure 7B). 2b of the germarium from ovaries of wMelPop-infected On the other hand, the increase in the number of ger- D. melanogasterw1118 was normal, thereby indicating that maria containing apoptotic cysts may result from the Wolbachia presumably did not negatively affect follicle action of the bacteria on the SSCs, which gives rise to fol- cells. It should be noted that the fecundity of the wMel- licle cells in region 2b of the germarium (Figure 7A, C). Pop infected D. melanogaster w1118 was not decreased as Drummond-Barbosa and Spradling [8] have suggested compared with their uninfected counterparts [43,44]. that apoptosis in region 2a/2b of the germarium serves to This was evidence of insect plasticity, rendering them maintain the proper ratio of germline cells to somatic fol- capable to adapt to diverse factors. licle cells. In poorly fed flies, follicle cells slow down their Taken together, our findings clearly demonstrated that proliferation, the germline cells to somatic follicle cell the Wolbachia strain wMelPop has an effect on the egg ratio becomes skewed, resulting in cyst apoptosis in chamber formation in the D. melanogaster germarium. region 2a/2b which corrects this ratio [8]. It has been However, the underlying mechanism is still unclear. We established that stem cells are maintained in specialized intend to perform a comparative morphometric analysis microenvironment called the niche [42]. The abundance of apoptotic structures and bacteria in cystocytes of of Wolbachia in the SSCN [26] is of interest in this con- wMel- and wMelPop-infected flies. The results would be text. Thus reasoning, it may be assumed that the pre- helpful in deciding whether the increase in apoptosis sence of Wolbachia in the SSCN decreases the SSC frequency is due to high bacterial density or to particu- proliferation rate, the ratio of germline cells to follicle lar pathogenic effect of the Wolbachia strain wMelPop cells becomes imbalanced and, as a consequence, cysts on female germline cells. Zhukova and Kiseleva BMC Microbiology 2012, 12(Suppl 1):S15 Page 9 of 12 http://www.biomedcentral.com/1471-2180/12/S1/S15

Figure 6 Ultrastructure of the germarium cells at the periphery of region 1 in wMelPop-infected D. melanogasterw1118. A, a fragment of region 1 of the germarium, low magnification view. Normal cells and two fragments of cells (brackets), whose cytoplasm is filled with autophagosomes, bacteria and multilayered membranes. B, multilayered membranes and fragments of a disintegrated nucleus (white arrowhead). C, a fragment of a cell with electron-dense cytoplasm containing Wolbachia of two types: one normal (black arrows), the other with matrix of low density (white arrows). D, electron-dense bacteria-like structures engulfed by autophagosome. E, higher magnification of the bacteria-like structure framed in panel D. F, an autophagosome containing electron-dense structures and vesicles . G, autophagosomes enclosed individual bacteria. Arrowheads indicate autophagosome membranes. Scale bars: 1 μm.

Conclusions Future research should be conducted to clarify the The results of this study showed that the presence of mechanism underlying this phenomenon. the Wolbachia strain wMelPop in D. melanogaster ovaries led to an increase in the frequency of apoptosis in Methods the germarium checkpoint. Two possible pathways Drosophila stocks and maintenance along which Wolbachia affect egg chamber formation in The Drosophila melanogaster Canton S infected with the region 2a/2b of the germarium have been suggested. Wolbachia strain wMel (IC&G, Russia) and D. Zhukova and Kiseleva BMC Microbiology 2012, 12(Suppl 1):S15 Page 10 of 12 http://www.biomedcentral.com/1471-2180/12/S1/S15

Figure 7 Pathways along which Wolbachia may affect egg chamber formation in region 2a/2b of the germarium. A, localization of regions in the germarium (framed) where the bacteria may interfere with normal function of cells. B, the bacteria disturb the differentiation of cystocytes (white) into the oocyte (light orange) and the nurse cells (light violet). C, the bacteria skew the proper ratio of germline cells to follicle cells. Crescent shape, SSCN; green circle, SSC; green ovals, follicle cells. Red points represent the bacteria.

w1118 melanogaster infected with wMelPop (a kind gift mM KCl, 1.9 mM CaCl2,10мM Hepes pH 6.9), stained from prof. S. O’Neill, The University of Queensland, with AO (Merck), 5 μg/ml, in 0.1 M sodium phosphate Australia) were used in these experiments. Flies were buffer, pH 7.2, for 3 min at room temperature [12,47]. maintained at 25 °C either on a standard yeast-agar Samples were placed onto glass slides and covered with medium or on daily replaced rich food (standard med- halocarbon oil (KMZ Chemicals Ltd.). They were viewed ium covered with wet yeast paste). To obtain uninfected under an Axioscop 2 plus fluorescence microscope D. melanogasterw1118T, flies were raised on food supple- (Zeiss) using an appropriate filter (Zeiss filter set 02). mented with tetracycline at 0.03% for two generations, Time elapsed from dissection to the end of viewing was then on standard food for more than three generations restricted, 20 min. Staining of nuclei varied from bright [43]. Confirmation of the infection status of each stock yellow to brilliant orange, depending on the stage of was provided by PCR. For this purpose, total DNA degeneration [46]. The percentage of AO-staining ger- extracted from fly ovaries and wsp 81F/wsp 691R pri- maria was expressed as the ratio of the number of AO- mers for amplifying a Wolbachia surface protein gene stained germaria containing apoptotic cells to the total fragment were used [45]. number of analysed germaria. Three experiments were performed for each of the 4 D. melanogaster groups Acridine orande staining (w1118, w1118T stocks, standard food; w1118, w1118T, Acridine orange (AO), a vital stain highly specific to rich food). In each replicate, ovaries were dissected from apoptotic nuclei, was used [46]. Ovaries were dissected 6 flies, 7-12 germaria per fly were analysed. In all, about from 5-day old flies in EBR buffer (130 mM NaCl, 4.7 1350 AO-stained germaria were analysed. Bartlett’stest Zhukova and Kiseleva BMC Microbiology 2012, 12(Suppl 1):S15 Page 11 of 12 http://www.biomedcentral.com/1471-2180/12/S1/S15

was used to check homogeneity of variances. Two-way distinct puncta in region 2a/2b of the germarium from the same fly ANOVA was used to determine the significance of the stocks, as in A, B. E, F, the TUNEL-positive germaria with clusters of bright difference between the frequency of apoptosis of the spots. Region 2a/2b of the germarium is indicated by red brackets. Scale μ uninfected and Wolbachia-infected flies maintained on bars: 20 m. Additional file 2: Cystocytes in region 2a/2b of the germarium from different food. the wMel-infected D. melanogaster Canton S. Bacteria with moderate density matrix are indicated by black arrows; white arrow points to a TUNEL assay bacterium with light matrix; ring canals between cystocytes are marked μ TUNEL was the independent assay of detection of apop- by arrowheads. Scale bar: 2 m. Additional file 3: Morphology of apoptotic cystocytes in region 2a/ totic cells. TUNEL is advantageous because preferen- 2b of the germaria from the uninfected D. melanogasterw1118T. A, tially labeling apoptotic cells relatively late in the cyst cells containing swollen mitochondria (arrows). B, a normal apoptotic process [48]. Ovaries were dissected from 5- mitochondrium (arrowhead) and swollen mitochondria in the cytoplasm of a cyst cell. C, pyknotic nuclei in cyst cells. D, an apoptotic body (ab) day old flies in phosphate-buffered saline (PBS), fixed in containing remnants of a fragmented cell. Scale bars: 1 μm. PBS containing 4% formaldehyde plus 0.1% Triton X- Additional file 4: The Wolbachia strain wMel in cyst cells 100 for 25 min. Then, they were separated into indivi- undergoing apoptosis in region 2a/2b of the germaria. A, apoptotic dual ovarioles, rinsed briefly in PBS twice and washed in cystocytes, low magnification view. B, bacteria framed in panel A depicted at higher magnification. Bacteria showing normal morphology PBS three times for 5 min each. Ovarioles were made (arrows), with light matrix (white arrowheads), with light matrix and permeable with 20 μg/ml proteinase K in PBS for 20 disrupted envelope (black arrowheads) in the cytoplasm of dying cell. min at room temperature, this was followed by 3 washes Scale bars: 2 μm. in PBS for 5 min each. The TUNEL reaction and all the Additional file 5: Follicle cells in region 2b of the germaria from wMelPop-infected D. melanogasterw1118. A, follicle cells containing subsequent steps were performed using the FragEL small amounts of bacteria (arrows). B, follicle cells and apoptotic cyst DNA Fragmentation Detection Kit (Calbiochem) accord- cells (ac). Scale bars: 2 μm. ing to the manufacturer’s protocol. Samples were viewed Additional file 6: Ultrastructure of germarium cells at periphery of with an Axioscop 2 plus fluorescent microscope (Zeiss), region 1 in wMel-infected D. melanogaster Canton S. A, B, fragments of cells whose cytoplasm contains numerous autophagosomes, bacteria images were captured with a high resolution microscopy and multilayered membranes (low magnification view). C, high- camera AxioCam HRc and AxioVision software. Ger- magnification micrograph of the fragment shown in panel A (framed) maria from ovaries of 10 flies were counted in each of demonstrating a bacterium enclosed by autophagosome. D-F, autophagosomes containing numerous membranes and inclusions the 4 groups. The total number of germaria analysed varying in electron density. Scale bars correspond to 1 μm (A, B) and 0.5 wasabout850.ThedatawerecomparedusingaChi- μm (C-F), respectively. square test (c2).

Electron microscopy Acknowledgements Fixation of the D. melanogaster ovaries was carried out We thank Prof. S. O’Neill (The University of Queensland, Australia) for kindly using the method described previously [49,35]. Briefly, 5 supplying us with D. melanogaster stock. We are also grateful to the staff of the IC&G SB RAS, particularly to Dr. A.A. Ogienko for sharing her experience day-old females were dissected in 0.1 M phosphate buf- with AO-staining of the D. melanogaster ovaries, Prof. I.K. Zakharov for fer, pH 7.4, fixed in 2.5% glutaraldehyde (Sigma) in 0.1 providing conditions for fly maintenance, A.N. Fadeeva for translating the M sodium cacodylate buffer, pH 7.4, for 2.5 h. This was manuscript from Russian into English. This work was supported by the Program of Basic Research of the RAS Presidium “Biodiversity” (26.30), followed by washings in the same buffer and postfixa- “Molecular and Cellular biology” (6.12) and a grant from the Russian tion in 1% OsO4 and 0.8% potassium ferrocyanide for 1 Foundation for Basic Research. h. After washings, samples were placed in 1% aqueous This article has been published as part of BMC Microbiology Volume 11 solution of uranyl acetate (Serva) for 12 h at 4 °C. Then Supplement 1, 2012: Arthropod symbioses: from fundamental studies to pest and disease mangement. The full contents of the supplement are available they were dehydrated in ethanol series and acetone, online at http://www.biomedcentral.com/1471-2180/12?issue=S1. finally samples were embedded in Agar 100 Resin (Agar ’ Scientific Ltd.). Ultra-thin sections were stained with Authors contributions MZ performed the experiments. EK and MZ both designed the study, uranyl acetate and Reynolds lead citrate. They were drafted and wrote the manuscript. Both authors have read and approved examined with a transmission electron microscope (JEM the final text.

100 SX, JEOL). The number of flies analysed in each of Competing interests the 4 groups was 8-12. The authors declare that they have no competing interests.

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